Human cytomegalovirus (HCMV) is a ubiquitous, usually benign virus that establishes lifelong persistence or latency. However, HCMV frequently contributes to rejection of organs or cells in transplant patients and causes systemic disease and developmental defects of the CNS in neonates. In causing disease, HCMV infects many different cell types including epithelial and endothelial, glial cells, fibroblasts and monocyte- macrophages. This broad topism is, in large part, defined by a capacity to enter many different cell types by distinct entry pathways involving different cellular entry mediators and different forms of glycoprotein gH/gL: gH/gL/UL128-131 and gH/gL/gO. The three overlapping aims of the proposed studies will markedly expand our understanding of how HCMV enters different cell types and has broad importance for the design of HCMV vaccines. Aim 1 will characterize assembly of the gH/gL/UL128-131 and gH/gL/gO complexes, incorporation into the virion envelope, functions in entry, and recognition by neutralizing antibodies. Before we can better comprehend how different forms of gH/gL function, we need to better understand: i) the assembly of gH/gL/UL128-131 and gH/gL/gO, ii) the relative quantities of the gH/gL complexes incorporated into virions and iii) how these proteins are recognized by neutralizing antibodies in humans affecting entry spread in vivo. To address this, we will purify different forms of gH/gL, test functional properties, produce monoclonal antibodies and mutant forms of the proteins and study in vitro assembly of complexes, levels of proteins in virions and reactivity with human antibodies. Aim 2 will investigate the structural and functional properties of complexes formed between HCMV gB and gH/gL We recently demonstrated that HCMV gB, the putative fusion protein, stably interacts with gH/gL, which we believe binds recpetors. This relates to models describing all herpesvirus fusion machineries, but there is no solid proof for gB-gH/gL interactions, yet this is fundamental to the models. To define the functional importance of HCMV gB-gH/gL interactions and map interacting domains, we will produce gH/gL mutants that do not bind gB and test whether mutants retain the capacity to bind receptors but not mediate fusion or entry. Aim 3 will seek to identify cellular proteins that promote HCMV entry into epithelial and endothelial cells. The HCMV receptors that have been defined to date, do not explain the importance of HCMV gH/gL/UL128-131, which is required for virus entry into epithelial and endothelial cells. Our studies will identify novel epithelial and endothel cell proteins that act a entry mediators and explain the requirement for gH/gL/UL128-131.